Urinary tract infection (UTI) is the second most common infection in humans after those involving the respiratory tract. This results not only in huge annual economic costs, but in decreased workforce productivity, and high patient morbidity. Most infections are caused by uropathogenic Escherichia coli (UPEC). Antibiotic treatment is generally effective for eradication of the infecting strain, however, documentation of increasing antibiotic resistance, allergic reaction to certain pharmaceuticals, alteration of norma gut flora, and failure to prevent recurrent infections, represent significant barriers to treatment As a result, approaches to prevent UTI such as vaccination represent a gap that must be addressed. The laboratory has an outstanding track record in the field of vaccine development and has made progress toward development of a preventive vaccine against UPEC. The long-term research goal is to prevent UTIs in women with recurrent UTI. The objective during this funding period is to identify the optimal combination of protective antigens for inclusion in an effective UTI vaccine, optimal adjuvant, optimal dose, and optimal route of delivery. The central hypothesis states that a multi-subunit vaccine elicits antibody or cell-mediated immunity that protects against experimental challenge with UPEC strains. The rationale for the proposed work is to protect women from the development of UTI by administration of a preventive vaccine. The central hypothesis will be tested and objectives will be completed by carrying out two specific aims: 1) Combine protective antigens to establish an effective multi- subunit vaccine to prevent urinary tract infection. 2) Identify the mechanism of protection for the optimized vaccine against uropathogenic E. coli in urinary tract infection. Previously, we systematically identified four antigens that can individually protect experimentally infected mice from colonization of the bladder and/or kidneys by UPEC when administered intranasally with cholera toxin (CT) as adjuvant. To advance the vaccine for utility in humans, we will group the individual antigens (IreA, Hma, IutA, FyuA) into an effective combination to establish a multi-subunit vaccine, and optimize the adjuvants, doses, and routes of inoculation currently used for immunization of humans. We demonstrated for all four vaccine antigens that antigen-specific serum IgG represents a strong correlate of protection in vaccinated mice. High antibody titers correlate with low CFUs of UPEC following transurethral challenge of vaccinated mice. The contribution of cell-mediated immunity cannot be ruled out and will also be investigated experimentally following vaccination. We propose to assess long-term protection, passive immunization, the involvement of B and T cells in protection, and whether antibodies bind to the surface of UPEC and inhibit iron acquisition and uptake. Sera from women with and without histories of UTI will be tested for antibody levels to vaccine antigens, epitope recognition, and the potential for eliciting opsonophagocytosis of UPEC. At the conclusion of the funding period, our expected result is an optimized vaccine ready for the next phase of development to prevent UTI in women with recurrent UTI.